Abstract The human immunodeficiency virus (HIV) integrase inhibitors are increasingly being used for antiretroviral therapy (ART), and dolutegravir (DTG) has emerged as a leading core agent. The DTG/Tivicay manufacturer reports (09/2018) that animal reproduction studies showed no evidence of adverse developmental outcomes, but an ongoing observational human cohort study in Botswana initially reported a 9-fold increase for neural tube defect (NTD) risk in offspring from mothers receiving DTG. With increased exposed births but no additional NTDs, a 6-fold increase for NTD risk in infants with early gestational exposure to DTG still remains. Recent concerns about teratogenicity have led to caution for DTG-based regimen use in women of child-bearing potential. We hypothesized that if DTG is teratogenic, then embryonic exposure to DTG will result in changes to one or more essential developmental processes, affecting functional mechanisms that have direct roles in neurulation and NTDs. We report a mechanism of action (MOA) for DTG teratogenicity and demonstrate specificity of this MOA in an animal model by rescue of DTG-induced developmental toxicity. Competitive binding data indicates DTG is a partial antagonist of folate receptors at clinically relevant concentrations. Data from the zebrafish model show developmental toxicity due to early embryonic exposure to DTG. Specificity of DTG developmental toxicity is demonstrated via rescue of DTG-induced developmental toxicity by supplemental folate. Folates and folate receptor are established modifiers of risk for NTDs, and these data indicate DTG is an antagonist of folate receptor and developmental toxicant at clinically relevant concentrations.